90 min 18 min. 45 min. 14 d

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1 Isolation, cultivation, and expansion of Pan T cells from human PBMCs In vitro expansion of human Pan T cells Introduction T lymphocytes (T cells) play a central role in the adaptive immune system by controlling a variety of cellular responses defending the host against pathogens and tumor development. For example, cytokine secretion by T helper cells suppresses or stimulates immune responses and leads to antibody production by B cells, isotype switching, and macrophage activation. Cytotoxic T cells however directly kill cancer cells, virus-infected cells, or otherwise damaged cells. Their crucial impact on immune responses and distinct role in the protection against disease make T cells a focus of many researchers studying immune regulation. It is all the more important to provide a reliable workflow for the isolation and cultivation of human Pan T cells directly from peripheral blood mononuclear cells (PBMCs) that is fully compatible with your downstream application of choice. Pan T cells are isolated directly from human PBMCs by magnetic enrichment using the Pan T Cell Isolation Kit, human and subsequently activated and expanded with the T cell Activation and Expansion Kit, human. T cell purity, proliferation, and expression of activation markers is assessed by flow cytometry at different time points. Workflow 90 min 18 min 45 min 14 d 8 min PBMC isolation Isolation of human Pan T cells Analysis of purity after cell separation CFSE labeling for analysis of cell proliferation Activation and expansion of human Pan T cells Flow cytometry analysis of activation markers (e.g. day 2) Analysis of cell proliferation and expansion rates (e.g. days 3, 6, 8, 10, 13, and 14) Removal of MACSiBead Particles (optional) In vitro expansion of human Pan T cells - 1 -

2 Materials Isolation of PBMCs using Ficoll-Paque PBS-EDTA: Phosphate-buffered saline (PBS), ph 7.2, with 2 mm EDTA. Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-a (ACD-A) or citrate phosphate dextrose (CPD). Ficoll-Paque (ρ = g/ml) 50 ml conical centrifuge tubes T cell cultivation TexMACS Medium (# ) Human IL-2 IS, premium grade (# ) 24-well plate (e.g. Gas-permeable Culture Plate # ) Note: With the Gas-permeable Culture Plate, up to cells/well/ml can be stimulated as opposed to PBMCs/well/mL in standard 24-well plates. Optional: RPMI 1640 medium 100 L-Glutamine stock solution (200 mm) 100 penicillin/streptomycin stock solution 2-Mercaptoethanol Fetal bovine serum (FBS) Buffer (standard wash and dilution buffer) automacs Rinsing Solution (# ) Bovine serum albumin (BSA Stock Solution; # ) Magnetic cell separation Pan T Cell Isolation Kit, human (# ) LS Columns (# ) MACS Separator for LS Columns (e.g. MidiMACS Separator; # ) MACS MultiStand (# ) CFSE labeling for analysis of cell proliferation 5(6)-Carboxyfluorescein diacetate N- succinimidyl ester (CFSE; MW , e.g. from Sigma-Aldrich; # MG-F) Dimethyl sulfoxide (DMSO; e.g. CryoMACS DMSO 10; # ) Phosphate-buffered saline (PBS), ph 7.2 Human AB serum T cell activation and expansion T Cell Activation/Expansion Kit, human (# ) Analysis of phenotypic and activation T cell markers Antibodies, human: CD2-PE (# ) CD3-FITC (# ) CD69-APC (# ) CD25-PE (# ) CD3-APC-Vio 770 (# ) MACSQuant Analyzer, MACSQuant Analyzer 10 (# ), or other flow cytometers equipped with violet (405 nm), blue (488 nm) and red (635 nm) lasers able to discriminate FITC, PE, and APC fluorescence Note: The MACSQuant VYB cannot be used. MACS Chill 96 Rack (# ), when using MACSQuant Analyzer or MACSQuant Analyzer 10 MACSQuant Calibration Beads (# ), when using the MACSQuant Analyzer or MACSQuant Analyzer 10 Removal of MACSiBead Particles MACSiMAG Separator (# ) In vitro expansion of human Pan T cells - 2 -

3 Material preparation T cell medium Make sure to add IL-2 freshly to the T cell medium for cell expansion. Instead of TexMACS Medium, RPMI supplemented with FBS (10% final concentration), 100 L-Glutamine stock solution (1% final concentration), 2- mercaptoethanol (0.01 mm final concentration) and human IL-2 (50 IU/mL) can be used for T cell cultivation and expansion. Addition of penicillin/streptomycin to the T cell medium is optional (e.g. 100 penicillin/streptomycin stock solution to a final concentration of 1%). Prepare TexMACS Medium supplemented with human IL-2 (50 IU/mL). CFSE stock solution for cell labeling Prepare the 10 mm CFSE stock solution by dissolving, e.g., 5.57 mg of CFSE (MW ) in 1 ml of DMSO. Use CFSE at a final concentration of 1 µm by diluting the stock solution 1:10,000 (e.g. 1 µl CFSE stock solution per 10 ml cell suspension in PBS). Aliquots of the stock solution should be stored at 20 C or below. Avoid repeated freeze-thaw cycles. Buffer (standard wash and dilution buffer) Prepare a solution of PBS, ph 7.2, 2 mm EDTA and 0.5% BSA by diluting MACS BSA Stock Solution 1:20 with automacs Rinsing Solution. Reconstitution of Human IL-2 IS, premium grade It is recommended to reconstitute lyophilized Human IL-2 IS, premium grade with deionized sterile-filtered water to a final concentration of mg/ml in a volume of at least 100 µl. Note: Further dilutions should be prepared with 0.1% BSA or human serum albumin (HSA) in PBS. The ED₅₀ is 0.2 ng/ml corresponding to a specific activity of IU/mg (calibrated with NIBSC 86/504) or 1 10⁷ IU/mg (calibrated with Proleukin ). Recommended stock concentration: 0.1 mg/ml by reconstituting a 10 µg vial of Human IL-2 IS, premium grade with 100 µl deionized sterile-filtered water. This results in a final activity of 500 IU/µL. Upon reconstitution aliquots should be stored at 20 C or below. Avoid repeated freeze-thaw cycles. To obtain a cell culture medium supplemented with 50 IU/mL, add 1.0 µl reconstituted Human IL-2 IS, premium grade freshly to 10 ml cell culture medium. Loading of Anti-Biotin MACSiBead Particles Resuspend Anti-Biotin MACSiBead Particles (contained in the T Cell Activation/ Expansion Kit) thoroughly by vortexing before use, to obtain a homogenous suspension. Anti-Biotin MACSiBead Particles are supplied without preservative. Remove aliquots under aseptic conditions. It is recommended to load Anti-Biotin MACSiBead Particles in batches of particles. Loaded AntiBiotin MACSiBead Particles are stable for up to 4 months when stored at 2 8 C. 1. Pipette 100 µl of CD2-Biotin, 100 µl CD3- Biotin and 100 µl CD28-Biotin into sealable 2 ml tube and mix well. Note: This antibody combination, with a final antibody concentration of 10 µg per antibody per 1 ml of loaded Anti-Biotin MACSiBead Particles, is optimized for achieving maximal T cell activation. 2. Resuspend Anti-Biotin MACSiBead Particles thoroughly by vortexing. 3. Remove 500 µl Anti-Biotin MACSiBead Particles ( AntiBiotin MACSiBead Particles) and add to antibody mix. 4. Add 200 µl buffer to adjust to a total volume of 1 ml. Note: Anti-Biotin MACSiBead Particles can be loaded in a flexible manner with biotinylated antibodies or ligands other than those supplied in the T Cell Activation/Expansion Kit, human. If desired, add other biotinylated antibodies or ligands at appropriate concentrations and adjust with buffer to a total volume of 1 ml, accordingly. In vitro expansion of human Pan T cells - 3 -

4 5. Incubate for 2 hours at 2 8 C under constant, gentle rotation by using the MACSmix Tube Rotator (# ) at approximately 4 rpm (slowest permanent run program). 6. The loaded Anti-Biotin MACSiBead Particles ( particles/ml) are now ready to use. Do not remove the loaded Anti-Biotin MACSiBead Particles from the antibody mix. Store at 2 8 C for up to 4 months. Antibody panel for phenotyping Freshly prepare the following master mix for each sample: 80 µl buffer 10 µl of each antibody: CD3-FITC CD2-PE Store master mix in the dark in the refrigerator (2 8 C) until use. Do not store for extended periods. Antibody panel for activation markers Freshly prepare the following master mix for each sample: 70 µl buffer 10 µl of each antibody: CD69-APC CD3-APC-Vio 770 CD25-PE Store master mix in the dark in the refrigerator (2 8 C) until use. Do not store for extended periods. Note: Highly activated T cells might down-regulate CD3 to some extent. Alternatively, CD4 and CD8 stainings (e.g. with CD4-VioBlue ; # and CD8-VioGreen ; # ) can be used to properly gate on T cell subpopulations. Methods 1. Isolation of human PBMCs using Ficoll- Paque The peripheral blood or buffy coat should not be older than 8 hours and supplemented with anticoagulants (e.g. heparin, EDTA, citrate, ACD-A, or citrate phosphate dextrose (CPD)). 1. Dilute cells with 2 4 the volume of PBS- EDTA. Note: The more diluted the blood sample, the better the purity of the mononuclear cells. 2. Carefully layer 35 ml of diluted cell suspension over 15 ml of Ficoll-Paque in a 50 ml conical tube. 3. Centrifuge at 400 g for minutes at 20 C in a swinging-bucket rotor without brake. 4. Aspirate the upper layer leaving the mononuclear cell layer (lymphocytes, monocytes, and thrombocytes) undisturbed at the interphase. 5. Carefully transfer the mononuclear cell layer to a new 50 ml conical tube. 6. Fill the conical tube with PBS-EDTA, mix, and centrifuge at 300 g for 10 minutes at 20 C. Carefully remove supernatant completely. 7. For removal of platelets, resuspend the cell pellet in 50 ml of PBS-EDTA and centrifuge at 200 g for minutes at 20 C. Carefully remove the supernatant completely. Note: This step will increase the purity of the target cells in the subsequent MACS Cell Separation. 8. Repeat step 7. Most of the platelets will remain in the supernatant upon centrifugation at 200 g. 9. Resuspend cell pellet in an appropriate amount of PBS-EDTA and proceed to magnetic labeling. Note: PBMCs may be stored in the refrigerator overnight in PBS containing 0.5% BSA or autologous serum. Do not store cells longer than one day in the refrigerator. Wash at least once before proceeding to magnetic labeling and resuspend cells in an appropriate buffer. For details see MACS Cell Separation Reagents data sheets. 2. Magnetic labeling Work fast, keep cells cold, and use precooled solutions (2 8 C). Volumes for magnetic labeling given below are for up to 10⁷ total cells. When working with fewer cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly. For optimal performance it is important to obtain a single-cell suspension before magnetic labeling. In vitro expansion of human Pan T cells - 4 -

5 1. Determine cell number of PBMCs. 2. Resuspend cell pellet in 40 µl of buffer per 10 7 total cells. 3. Add 10 µl of Pan T Cell Biotin-Antibody Cocktail (contained in the Pan T Cell Isolation Kit, human) per 10 7 total cells. 4. Mix well and incubate for 5 minutes in the refrigerator (2 8 C). 5. Add 30 µl of buffer per 10 7 total cells. 6. Add 20 µl of Pan T Cell MicroBead Cocktail per 10 7 total cells. 7. Mix well and incubate for 10 minutes in the refrigerator (2 8 C). 8. Proceed to subsequent magnetic cell separation. Note: A minimum of 500 µl is required for magnetic separation. If necessary, add buffer to the cell suspension. 3. Magnetic separation Always wait until the column reservoir is empty before proceeding to the next step. Choose an LS Column and a suitable MACS Separator. 1. Place LS Column in the magnetic field of a suitable MACS Separator. For details refer to the respective MACS Column data sheet. 2. Prepare column by rinsing with 3 ml of buffer. 3. Apply cell suspension onto the column. Collect flow-through containing unlabeled cells, representing the enriched T cells. 4. Wash column with 3 ml of buffer. Collect unlabeled cells that pass through, representing the enriched T cells, and combine with the effluent from step (Optional) Remove column from the separator and place it on a suitable collection tube. Pipette 5 ml of buffer onto the column. Immediately flush out the magnetically labeled non-t cells by firmly pushing the plunger into the column. 4. Analysis of purity after cell separation This step is optional. Additional antibodies can be included in the analysis according to the respective needs. For additional antibodies visit 1. Remove a small aliquot from the fraction representing the enriched T cells (e.g. 50 µl). 2. Determine cell number. 3. Centrifuge cell suspension at 300 g for 10 minutes. Aspirate supernatant completely. 4. For each sample resuspend up to 10 6 nucleated cells in 100 µl phenotyping master mix (see Material preparation, section Antibody panel for phenotyping). 5. Mix well and incubate for 10 minutes in the dark in the refrigerator (2 8 C). Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times. 6. Wash cells by adding 1 2 ml of buffer and centrifuge at 300 g for 10 minutes. Aspirate supernatant completely. 7. Resuspend cell pellet in a suitable amount of buffer (e.g. 500 µl) for analysis by flow cytometry. Note: Add propidium iodide according to the manufacturer s instructions before flow analysis. 5. CFSE labeling for analysis of cell proliferation CFSE is added to the freshly isolated Pan T cell suspension to assess cell proliferation at any given time point and for every condition tested. Therefore, please do not use antibodies for flow analysis conjugated to fluorochromes that exhibit the same spectrum as CFSE (λ ex 492 nm; λ em 517 nm). Alternatively, a separate well in the cell culture dish can be used for CFSE labeling. Please make sure to prepare at least one additional well per In vitro expansion of human Pan T cells - 5 -

6 condition (e.g. stimulated and non-stimulated control). Cell proliferation is usually analyzed on days 6, 8, and 10. However, please feel free to choose time points that are appropriate for your experimental needs (see section 8. Analysis of cell proliferation and expansion rates). To assess expansion rates, please also determine cell numbers, e.g., on days 3, 6, 8, 10, 13, and 14 using, e.g., a Neubauer Chamber or the counting function of the MACSQuant Analyzer 10 (see section 8. Analysis of cell proliferation and expansion rates). 1. Determine cell number. 2. Centrifuge cell suspension at 300 g for 10 minutes. Aspirate supernatant completely. 3. Resuspend cells to a final concentration of cells/ml in PBS. 4. Add the CFSE stock solution to a final concentration of 1 µm to the cell suspension (e.g. add 1 µl 10 mm CFSE stock solution to 10 ml cell suspension). 5. Mix well and incubate for 10 minutes at 37 C. 6. Add one volume of human AB serum, mix well and incubate for 5 minutes at room temperature (e.g. add 10 ml human AB serum to 10 ml cell suspension). 7. Centrifuge cell suspension at 300 g for 10 minutes. Aspirate supernatant completely. 8. Resuspend cells to a final concentration of cells/ml in TexMACS Medium without supplements. 9. Repeat steps 7 and 8 to wash cells twice. Note: After the last wash resuspend cells in fresh TexMACS Medium supplemented with IL Cells are now ready for activation and expansion. Proceed to subsequent step. 6. T cell cultivation, activation, and expansion with MACSiBead Particles 1. Resuspend loaded Anti-Biotin MACSiBead Particles thoroughly and transfer 10 μl (1 10⁶ loaded Anti-Biotin MACSiBead Particles) per 2 10⁶ cells into a suitable tube. Note: If unloaded MACSiBead Particles will be used for negative control experiments, replace the loaded Anti- Biotin MACSiBead Particles with 10 μl (1 10⁶ beads) of unloaded Anti-Biotin MACSiBead Particles per 2 10⁶ cells. 2. Add µl of TexMACS Medium without supplements to the loaded Anti-Biotin MACSiBead Particles and centrifuge at 300 g for 5 minutes. 3. Aspirate supernatant and resuspend loaded Anti-Biotin MACSiBead Particles in 100 µl of fresh TexMACS Medium supplemented with IL Resuspend cells at a density of 2 10⁶ cells per 900 µl of TexMACS Medium supplemented with IL Add the prepared Anti-Biotin MACSiBead Particles from step 3 to the 900 µl of cell suspension and mix well. 6. Dilute cells with TexMACS Medium supplemented with IL-2 to a final density of cells per ml per cm 2 and add the mixture to a suitable cell culture vessel (e.g. 2 10⁶ cells in 2 ml per well of a 24-well plate). 7. Incubate at 37 C and 5 10% CO₂ for up to 3 days. Note: Inspect cultures daily, and add fresh TexMACS Medium supplemented with IL-2 if required. 8. At day 3 gently pipette cell suspension up and down to break up clumps. 9. Split cell suspension into two equal parts and add TexMACS Medium supplemented with IL-2. Incubate at 37 C and 5 10% CO Split cell suspension again whenever necessary (e.g. every 2 3 days or when cells reach 80% confluency) into two equal parts and add TexMACS Medium supplemented with IL-2. Incubate at 37 C, 5 10% CO 2. In vitro expansion of human Pan T cells - 6 -

7 Note: The ideal starting cell density for T cell expansion is T cells per ml. Inspect the cell culture daily. Depending on the expansion rate, it might be necessary to split the culture more frequently than every 2 3 days. If IL-2 interferes with downstream experiments, it can be omitted. However, omission will lower the cell viability. 11. At day 14, resuspend T cells at cells per ml in fresh TexMACS Medium supplemented with IL For longer expansion periods, restimulate the cells by adding one loaded Anti-Biotin MACSiBead Particle per two cells To this end, count the cells and add 12.5 μl loaded Anti- Biotin MACSiBead Particles per T cells. 13. Further cultivate cells and repeat steps 8 and 9 every 2 3 days. Note: Inspect the cultures daily. Depending on the expansion rate, it might be necessary to split cultures more frequently than every 2 3 days. 14. Proceed to downstream application, e.g. analysis of cells. Note: Removal of Anti-Biotin MACSiBead Particles is not required for immunofluorescent staining. For assays where T cells are required to return to a fully resting state prior to further stimulation, Anti-Biotin MACSiBead Particles should be removed at least 24 hours before restimulation (see section 9. Removal of MACSiBead Particles). 7. Flow cytometry analysis of activation markers For analysis of T cell activation, samples are taken 48 hours after stimulation and stained for early activation markers CD25 and CD69 to determine the proportion of activated cells among viable CD3 + T cells. Highly activated T cells might down-regulate CD3 to some extent. Alternatively, CD4 and CD8 stainings (e.g. with CD4-VioBlue ; # and CD8-VioGreen ; # ) can be used to properly gate on T cell subpopulations. Additional antibodies can be included in the analysis according to the respective needs. This specific panel is compatible with CFSE labeling. When modifying the antibody panel, make sure to use fluorochrome-conjugated antibodies that do not interfere with CFSE analysis (see section 5. CFSE labeling for analysis of cell proliferation) or use separate wells for CFSE labeling. For additional antibodies visit 1. Remove a small aliquot from samples (e.g. stimulated sample and unstimulated control). 2. Determine cell number. 3. Centrifuge cell suspension at 300 g for 10 minutes. Aspirate supernatant completely. 4. For each sample resuspend up to 10 6 nucleated in 100 µl phenotyping master mix (see Material preparation, section Antibody panel for phenotyping). 5. Mix well and incubate for 10 minutes in the dark in the refrigerator (2 8 C). Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times. 6. Wash cells by adding 1 2 ml of buffer and centrifuge at 300 g for 10 minutes. Aspirate supernatant completely. 7. Resuspend cells in a suitable amount of buffer (e.g. 500 µl) for analysis by flow cytometry. Note: Add propidium iodide according to the manufacturer s instructions before flow analysis. 8. Analysis of cell proliferation and expansion rates 1. For monitoring cell proliferation samples of µl are taken at appropriate time points (e.g. days 6, 8, and 10), diluted 1:2 with buffer, and CFSE dilution is measured on the MACSQuant Analyzer 10. At least 20,000 events are recorded. Note: Time points can be modified to meet experimental needs. 2. For determination of the cell expansion rate samples are taken at appropriate time points (e.g. days 3, 6, 8, 10, 13, 14) and the count of viable cells is measured on the MACSQuant Analyzer 10. For death cell exclusion PI is added prior to flow cytometric acquisition. Expansion rate is calculated afterwards. In vitro expansion of human Pan T cells - 7 -

8 9. Removal of MACSiBead Particles (optional) For some experiments (e.g. restimulation of T cells) it is recommended to remove the MACSiBead Particles from the cell suspension. 1. Harvest cells and pool cells from wells that were treated under the same conditions. Wash empty wells with cold buffer to rinse out the remaining cells on the plate. 2. Determine cell number. 3. Wash cells with cold buffer. Figure 1: Flow cytometry analysis of T cell purity after magnetic separation. Separation of untouched T cells from human PBMCs by depletion of non-t cells using the Pan T Cell Isolation Kit (human), an LS Column, and a MidiMACS Separator. Cells were labeled with CD2-PE and CD3-FITC and analyzed by flow cytometry using the MACSQuant Analyzer. 4. Resuspend cells in buffer at a density of up to /ml and vortex thoroughly. 5. Place the tube in the magnetic field of the MACSiMAG Separator. 6. Allow the MACSiBead Particles to adhere to the wall of the tube for 4 minutes. 7. With the tube still placed in the MACSiMAG Separator, carefully remove the supernatant containing the cells depleted of MACSiBead Particles. Transfer cells to a new tube. 8. Remove the tube from the separator and add the same volume of buffer as before. 9. Vortex sample, place tube in the MACSiMAG Separator and repeat steps Collected cells can now be further processed as required. Figure 2: Flow cytometry analysis of activation markers of human Pan T cells. (A) Pan T cells were isolated from human PBMCs using the Pan T Cell Isolation Kit, human. Cells were fluorescently stained with CD3, CD25, and CD69 antibodies 48 hours after activation using the T Cell Activation/ Expansion Kit, human (TCAE) and TexMACS Medium. Non-stimulated cells served as a control. (A) Flow cytometry analysis of CD25 and CD69 performed on the MACSQuant Analyzer 10. (B) Frequencies of CD69 + and CD25 + CD69 + Pan T cells with and without stimulation using the T Cell Activation/Expansion Kit, human (n = 9). In vitro expansion of human Pan T cells - 8 -

9 Figure 3: Analysis of cell proliferation. (A) Cell proliferation of human Pan T cells was analyzed by CFSE labeling at days 6, 8, and 10 after activation with the T Cell Activation/Expansion Kit, human (TCAE). CFSE dilution was determined using the MACSQuant Analyzer 10. Gray: cells stimulated with TCAE; black: non-stimulated control. (B) Proliferation rate 6 days after activation with the T Cell Activation/Expansion Kit, human (n = 9). Figure 4: Analysis of cell expansion. For the determination of the expansion rate, samples were taken at the time-points indicated and the count of viable cells was measured with the MACSQuant Analyzer 10. For death cell exclusion, PI was added prior to flow cytometric acquisition. Expansion rate was calculated afterwards. Miltenyi Biotec provides products and services worldwide. Visit to find your nearest Miltenyi Biotec contact. Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. automacs, CryoMACS, MACS, the MACS logo, MACSiMAG, MACSiBead, MACSmix, MACSQuant, MidiMACS, TexMACS, Vio, VioBlue, and VioGreen are registered trademarks or trademarks of Miltenyi Biotec GmbH and/or its affiliates in various countries worldwide. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only. Copyright Ó 2017 Miltenyi Biotec GmbH and/or its affiliates. All rights reserved. In vitro expansion of human Pan T cells - 9 -